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Transforming growth factor p expression in human marrow stromal cells Nemunaitis J, Tompkins CK, Andrews DF, Singer JW. Transforming growth factor P expression in human marrow stromal cells. Eur J Haematol 1991: 46: 140-145. Abstract: The effects of hematopoietic cytokines on the expression of transforming growth factors (TGFP) mRNA and the effect of TGFP on cytokine and on a major extracellular matrix protein, collagen I, mRNA expression was studied in human marrow stromal cells. As with other cultured mesenchymal cells, stromal cells constitutively express TGFP, but not TGFcl mRNA. In simian virus 40 (SV40)-transformed stromal cells downregulation of TGFP, expression was observed 2 hours after incubation with recombinant human (rh) tumor-necrosis factor cl (TNFcl) and 144 h after addition of rh granulocyte macrophage colony-stimulating factor (GM-CSF). Neither interleukin-l(IL-1)P nor IL-6 had an observable effect on TGFP, mRNA expression. TGFP upregulated collagen I mRNA expression. These data suggest that cytokines may influence TGFP mRNA expression.

Transforming growth factors a and P (TGFa and TGFP) are expressed in mesenchymal cells during early fetal development (1,2). TGFa has little effect on hematopoietic cell proliferation and is not expressed by unstimulated hematopoietic cells (3, 4). In contrast, TGFP is expressed in virtually all cells and may be a critical regulator of hematopoiesis (1, 5). It inhibits differentiation and proliferation of hematopoietic and other mesenchymal cells (5). For instance, TGFP inhibits cytokine-stimulated multipotent and committed progenitor colony formation and inhibits monocyte proliferation (6-8). Furthermore, TGFP blocks IL-Zdependent T-lymphocyte proliferation, B-lymphocyte differentiation and proliferation of myeloblastic leukemia cells (9-1 1). TGFP is also a potent inducer of several fibroblast extracellular matrix proteins including collagen types I, 111, V, sulfated proteoglycans and fibronectin (1, 5). The extracellular matrix proteins induced by TGFP also appear to play a significant role in the regulation of hematopoiesis (12-15). Marrow stromal cells are a heterogeneous population of cells which are the in vitro counterpart to the in vivo hematopoietic microenvironment. They are essential for sustained hematopoiesis in long-term marrow cultures in part mediated by production of Supported by Grants No. HL31782, CA 18029,CA 10333,CA 45672, CA 47748, and CA 16448 from the National Institutes of

Health, Bethesda, MD, and by research funds from the Veterans Administration. D.F.A. holds a Clinical Investigator Award from the National Cancer Institute.

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J. Nemunaitis, C. K. Tompkins, D. F. Andrews and J. W. Singer Medical Service, VA Medical Center, the Department of Medicine, Division of Oncology, the University of Washington, and the Fred Hutchinson Cancer Research Center, Seattle, Washington, U S A .

Key words: TGFP - GM-CSF - TNFcl - marrow stromal cells - SV40 - extracellular matrix Correspondence: John Nemunaitis, M.D., 11 I-Onc, VAMC, 1660 S, Columbian Way, Seattle, WA 98108, (2061 764-2524, FAX 206-764-2659 Accepted for publication 27 September 1990

extracellular proteins and proteoglycans (16-18). In order to assess the effects of TGFP on marrow stromal cells and to examine the expression of TGFP mRNA while avoiding analysis of heterogeneous populations of cells, cloned populations of human stromal cells were developed by transforming longterm marrow culture (LTMC)-adherent cells with simian virus 40 (SV40) (19, 20). Transformed marrow stromal cells form anchorage-independent colonies in semi-solid medium in response to either endogenously produced or exogenous cytokines (21). Unlike the parental stromal cell lines, the cell lines derived from colonies constitutively express mRNAs and proteins including G-CSF IL-lP, TNFa, IL-6, G-CSF, and GM-CSF. Futhermore, collagen I and fibronectin expression are significantly reduced in colony-derived stromal cells in comparison to the parental cell lines (21). Cytokines including G-CSF, GM-CSF, IL-1P and IL-6 were induced when parental stromal cell lines were treated with IL-1 (P or a), or TNFa (22). Concurrently, collagen I expression was markedly downregulated (23). Similarly, when either transformed or passaged, non-transformed marrow stromal cells were exposed to the DNA demethylating agent 5-azacytidineYupregulation of IL-6 mRNA w a s observed along with downregulation of collagen I mRNA (24). In the present study, we report the effects of cytokines on stromal cell TGFP mRNA expression as well as the effect of exogenous TGFP on cytolkine and collagen I mRNA expression. A related observa-

Regulation of TGFP tion of the effect of therapeutic administration of rhGM-CSF on TGFP, mRNA expression by mononuclear cells is also described. Material and methods Subjects

Long-term marrow cultures (LTMC) were generated from marrow aspirates taken from normal marrow donors after written formal consent was obtained under protocols approved by the Institutional Review Board of the Fred Hutchinson Cancer Research Center. Cell cultures

LTMC were grown by a modification of the method of Gartner & Kaplan (25). Adherent stromal cells from LTMC were treated with trypsin in EDTA and passaged 1 to 2 times before they were used either for direct studies or for transformation. SV40 was used to transform adherent stromal cells as described (19). Briefly, adherent cells from LTMC were reseeded into fresh flasks and exposed to 10 particles/cell of wild-type SV40 (Vero7, Meloy Laboratories, Springfield,Virginia). After several weeks, the transformation became apparent with an increase in the growth rate and a loss of contact inhibition. The transformed stromal cell cultures were maintained in McCoy's 5A medium with 10% fetal calf serum and were passaged weekly. To test the effect of various cytokines on TGFP, expression, the cells were passaged into 150cm3 flasks and the cytokines to be tested were added at concentrations previously shown to be active (21). The time course experiments were performed in such a manner that all flasks were harvested at the same time to minimize the effect of differing degrees of confluence. The cytokines tested for effect on TGFP, expression included the following: rhIL-6 as conditioned medium from yeast (specific activity = 5 x lo4 unitsfmg), rhIL-la (specific activity = 1.3 x lo7 units/mg) and rhGM-CSF (specific activity = 5. x lo7 units/mg) all kindly donated by Immunex Corporation, Seattle, Washington. RhTNFa (specific activity = 4 x lo7 units/mg) was kindly donated by Genentech, San Francisco, California. TGFP (specific activity = 1 x lo6 unitslmg) was purchased from Collaborative Research Inc., Bedford, Massachusetts. Mononuclear cell sample

Blood (100 ml) was drawn from a patient (unique patient number 493 1) 16 days and 46 d after matched allogeneic bone marrow transplant. The patient had received rhGM-CSF (250 pg/m2/d by 2-h intra-

venous infusion; specific activity 5 x lo7 colony forming units/mg; supplied by Immunex Corporation, Seattle, Washington) from d 0 to 16. Mononuclear cells were separated at room temperature using differential centrifugation separation with lymphocyte separation medium (Organon Teknika, North Carolina). Cells were washed two times with Hanks balanced salt solution (Gibco, NY) then three times with 1 x standard saline citrate and pelleted. RNA extraction

RNA was extracted (21) as previously described from adherent cells from LTMC, from unstimulated SV40-transformed stromal cells, from transformed stromal cell lines derived from autonomously growing colonies, from peripheral blood mononuclear cells, and from transformed stromal cells stimulated with IL-la, IL-6, GM-CSF, TNFa and TGFP. The concentration of RNA was estimated by determining the absorbance at 260 nm. The quality of whole cellular RNA was confirmed by running 5 pg on a denaturing 1% agarose-formaldehyde gel and then checking for the presence of 28 S and 18 S ribosomal fragments by ethidium bromide staining. Northern blot analyses

Whole cellular RNA (10 pg per well) was run in a 1.O % denaturing agarose-formaldehyde gel. The resulting Northern blots were hybridized with fulllength cDNA probes for IL-6, GM-CSF, IL-1P and a 250-base pair G-CSF-specific fragment (Immunex Corporation, Seattle, WA), TNFa, TGFa and a 1050-basepair TGFP,-specific fragment (Genentech Corporation, San Francisco, California) and a 1.8 kb procollagen I fragment isolated from Hf677 (21). All cDNA's and fragments were labeled by random priming with P32CTP and hybridized under stringent conditions (26). To further ensure loading equivalence, the blots were hybridized with a chick beta actin probe. ResuIts TGFP, RNA expression in stromal cells

When Northern blots of whole RNA from three independently-derivedtransformed stromal cell lines were hybridized with a TGFP cDNA probe, TGFP mRNA was found to be constitutively expressed. TGFP, expression was upregulated by exogenous rhTGFP (10 ng/ml) within 24 H (Fig. 1). Neither IL-6 (500 u/ml)nor IL-lP (10 u/ml or 100 u/ml) had observable effects on TGFP, RNA expression when assessed over time intervals from 112 h to 144 h (data not shown). However, in three separate experiments

,

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Fig. 1 . Northern blot of whole cellular RNA from transformed stromal cells 24 h after rhTGFP (long/ml) was added (lane A) compared to control cells (lane B) hybridized with a TGFP, cDNA probe. The same blot probed with P actin cDNA is shown below.

Fig. 2. Northern blot of whole cellular RNA derived from transformed stromal cells after exposure to 100 u/ml of rhTNFu. The blot was hybridized with a TGFP, cDNA probe. The same blot probed with actin is shown below.

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Fig. 3. Northern blot ofwhole cellular RNA derived from transformed stromal cells exposed to 10,000 u/ml ofrhGM-CSF. The blot was hybridized with a TGFP, cDNA probe. Shown below is the same blot hybridized with an actin probe.

Fig. 4 . Northern blot of RNA derived from mononuclear cells of a patient while receiving rhGM-CSF (A) and 30 d after discontinuation of rhGM-CSF (B). The blots were hybridized with TGFP, (upper panel) and actin (lower panel) cDNA probes.

Regulation of TGFP

Fig. 6. Northern blot of RNA derived from transformed stromal cells at various time points after exposure to TGFP (10 ng/mg) hybridized with a pro-collagen I cDNA probe. Equivalence of loading and quality of RNA is shown below by ethidium bromide staining.

Fig. 5. Northern blot of RNA derived from colony-derived

transformed stromal cells (lane A) compared to parental transformed stromal cells from which they derived 6 weeks earlier (lane B). The cells were not stimulated with exogenous growth factors. The blots were hybridized with TGFP, (upper panel), ILlp (middle panel) and actin (lower panel) cDNA probes.

with independently-derived stromal cell lines, rhTNFa (10, 100 and 1000 u/ml) and rhGM-CSF (1000, 10000 u/ml) significantly down-regulated TGFP, expression. Down-regulation of TGFP, was maximal at 2 h and still observable at 48 h after rhTNFcr was added (see Fig. 2). In contrast, the downregulation of TGFP, with rhGM-CSF was not detectable until 48 h and was maximal after 144 h (see Fig. 3). Digital image analysis of the blots revealed that TGFP, expression was downregulated by 66% 144 h after rhGM-CSF was added and by 88% 2 h after addition of rhTNFcl (data not shown). RhGM-CSF did not induce expression of TNFa mRNA in the transformed stromal cells (data not shown). To determine if rhGM-CSF administered in vivo might have a similar effect on TGFP expression, RNA from blood mononuclear cells from an allogeneic bone marrow transplant patient (UPN 493 1) during and after a 16-d trial of rhGM-CSF was analyzed. Mononuclear cell mRNA expression of TGFP, was substantially less while UPN 493 1 was receiving rhGM-CSF therapy when compared to a

sample obtained while she was clinically stable 30 d after discontinuation of rhGM-CSF (see Fig. 4). TGFP, mRNA expression in stromal cell lines with constitutive cytokine expression

Previous studies (21) have shown that stromal cell lines derived from colonies constitutively express G-CSF, GM-CSF, IL-1P, TNFa and IL-6 mRNA and proteins. When the Northern blots from colonyderived cell lines were probed for TGFP its expression was less than that of the parental stromal cell lines (see Fig. 5 ) .

,,

Effect of TGFP on collagen 1 mRNA expression in transformed marrow stromal cells

When TGFP was added at concentrations of 1 and 10 ng/ml to colony-derived stromal cell lines, it had no effect on transformed stromal cell RNA expression of GM-CSF or TGFa when cells were harvested from 'I2 h to 72 h later. However, collagen I expression was significantly upregulated within 10 h (see Fig. 6). TGFa RNA expression

Whole RNAs extracted from unstimulated, nontransformed and transformed stromal cells were probed with a random primed cDNA probe for TGFa. In four separate experiments no expression of TGFcl was detected. RNA from a melanoma 143

Nemunaitis et al. tumor cell line, A375 (American Type Culture Collection, Rockville, Maryland) served as a positive control. RhIL-1P (10 u/cc, 100 u/cc), rhTNFa (10 u/cc, 100 u/cc, 1000 u/cc), rhIL-6 (500 u/cc), or rhGM-CSF (1000 u/cc, 10,000 u/cc) over various time points ranging from 112 h to 144 h failed to induce TGFa mRNA expression in transformed stromal cells (data not shown). Furthermore, colonyderived transformed stromal cells which constitutively expressed GM-CSF, G-CSF, M-CSF, ILlP, TNFa, TGFP,, and IL-6 mRNA did not express mRNA for TGFa by Northern blot analysis (data not shown). Discussion

Virtually all cells express TGFP, mRNA, (1, 9); however, data regarding the regulation of TGFP, expression is sparse. Pfeilschifter et al. have shown that calcitonin decreases TGFP protein production in cultured rat calvaria; however, mRNA analysis was not performed (27). The current data demonstrate that rhTNFa rapidly downregulates TGFP, mRNA expression while rhGM-CSF does so only after 48 h. These data suggest that the two cytokines may affect TGFP through disparate mechanisms. The physiologic relevance of the rhGMCSF effect is suggested by the level of mRNA expression in mononuclear cells from a patient who received rhGM-CSF. An additional reverse correlation between the presence of GM-CSF and TGFP is suggested by data from transformed colony-derived stromal cell lines which constitutively produce both TNFa and GM-CSF. They have decreased levels of TGFP, mRNA when compared to the parental stromal cell lines which do not produce either TNFa or GM-CSF (21). It is unlikely that rhGM-CSF downregulated TGFP expression by inducing TNFa since mRNA expression of TNFa was not detectable after stimulation of stromal cells with rhGM-CSF. The upregulation of TGFP, mRNA by TGFP observed in this study has been observed by others in fibroblasts and tumor cell lines (28). TGFP, mRNA expression was not enhanced and TGFa expression was not induced by rhIL-1, rhIL-6, rhTNFa, or rhGM-CSF. Others have identified upregulation of TGFP, mRNA in NRK fibroblasts after exposure to platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) (28). TGFP activity has also been shown to be increased by parathyroid hormone, 1,25-dihydroxyvitamin D, and IL-1 in other cell populations (27). Constitutive TGFa expression occurs in many non-hematopoietic, neoplastic cell lines, some transformed cell lines, normal human keratinocytes and in cells during early fetal development. However, inducing factors have not been identified (3, 4, 29). 144

TGFP is a potent stimulator of extracellular matrix production (5). It increases production of fibronectin, collagen type I, collagen type 111, laminin and sulfated proteoglycans (1, 5). Furthermore, the transcription of proteins which degrade the extracellular matrix (metalloprotease, collagenase, and stremelysin) can be blocked by TGFP (30). We observed upregulation of collagen I mRNA (a major matrix protein) in response to TGFP. Matrix molecules may have a significant role in hematopoietic cell proliferation and differentiation (12-15). TGFP’s modulatory effects on hematopoiesis may be mediated in part by control of matrix proteins as well as by direct effects on hematopoietic cells (6-1 1). References 1. ROBERTSAB, SPORNMB. Transforming growth factor p. Adv Cancer Res 1988: 51: 107-145. R. Transforming growth factor-cr: Structure and 2. DERYNCK biological activities. J Cell Biochem 1986: 32: 293-304. AB, WINKLERME, DERYNCK R. Transforming 3. SCHREIBER

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expression of c-abl protooncogene mRNA in cultured human marrow stromal cells. Proc Natl Acad Sci USA 1989: 86: 6788-6792. DY, LILLYMB, 23. NEMUNAITIS J, ANDREWS DF, MOCHIZUKI SINGERJW. Human marrow stromal cells: Response to interleukin-6 (IL-6) and control of IL-6 expression. Blood 1989: 74: 1929-1935. 24. ANDREWS DF, NEMUNAITIS JJ, TOMPKINS C, SINGERJW. The effect of 5-azacytidine on gene expression in marrow stromal cells. Mol Cell Biol 1989: 9: 2748-2751. 25. GARTNER SM, KAPLANHS. Long term culture of human bone marrow cells. Proc Natl Acad Sci USA 1980: 77: 4756. 26. FEINBERG AP, VOGELSTEINB. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983: 132: 6-13. 27. PFEILSCHIFTER J, MUNDYGR. Modulation of type p transforming growth factor activity in bone cultures by osteotropic hormones. Proc Natl Acad Sci USA 1987: 84: 2024-2028. 28. VAN OBBERGHEN-SCHILLING E, ROCHE NS, FLANDERS KC, SPORNMB, ROBERTSAB. Transforming growth factor p l positively regulates its own expression in normal and transformed cells. J Biol Chem 1988: 266: 7741-7746. 29. COFFEYRJ Jr, DERYNCK R, WILCOXJN et al. Production and auto-induction of transforming growth factor-a in human keratinocytes. Nature 1987: 328: 817-820. 30. EDWARDSDR, MURPHYG , REYNOLDSJJ etal. Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor. EMBO J 1987: 6: 1899-1904.

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